HIV is the causative agent of acquired immunodeficiency syndrome (AIDS). The trans-activation region (TAR) and the Rev-response element (RRE) (Rosen et al., 1988; Dayton et al., 1989; Malim et al., 1990) of HIV are found in unspliced or partially spliced HIV mRNA introns. During replication of HIV, the RRE and TAR RNAs interact with specific HIV proteins. The RRE is recognised by the HIV protein Rev (Daly et al., 1989; Zapp & Green, 1989; Cochrane et al., 1990; Heaphy et al., 1990; Malim et al., 1990) which stimulates mRNA export from the nucleus (Emerman et al., 1989; Malim et al., 1990; Malim & Cullen 1993; Fischer et al., 1994; Meyer & Malim, 1994; Bogerd et al., 1995; Stutz et al., 1995) via the formation of a Rev/RRE complex which displays a nuclear export signal that is essential for Rev-mediated export of RNA from the nucleus and also for Rev shuttling (Malim et al., 1991; Fischer et al., 1994; Meyer & Malim, 1994; Fischer et al., 1995; Stutz et al., 1995; Wen et al., 1995; Wolff et al., 1995). The Rev/RRE interaction regulates the cytoplasmic accumulation of HIV genomic and structural mRNAs and is therefore essential if the virus is to propagate.
The RRE contains a series of stem-loop structures protruding from a long central stem, known as Stem I (Dayton et al., 1989; Malim et al., 1989b; Dayton et al., 1992; Mann et al., 1994), as shown in FIG. 1 (RRE-WT; SEQ ID NO: 1). At the base of Stem IIb is a high-affinity Rev-binding motif which is recognised by a single Rev protein with a K.sub.d of approximately 1 nM (Bartel et al., 1991; Heaphy et al., 1991; Iwai et al., 1992; Kjems et al., 1992; Tiley et al., 1992). This high-affinity motif is a purine-rich bubble stabilised by non-Watson-Crick G.circle-solid.A and G.circle-solid.G base pairs (Heaphy et al., 1991; Bartel et al., 1991; Iwai et al., 1992; Pritchard et al., 1994). Together with a bulged-out uridine nucleotide, these non-Watson-Crick base pairs open the major groove of the mRNA duplex and permit the recognition of functional groups on the two base pairs either side of the bulged region inside the widened major groove. In addition to these base-specific contacts, phosphate contacts are made around the bubble as well as with base-paired nucleotides further away from the bubble (Iwai et al., 1992; Kjems et al., 1992; Pritchard et al., 1994).
Mutational analysis of the RRE has shown that the high-affinity interaction with a single Rev protein is necessary, but not sufficient, for Rev activity in vivo (Dayton et al., 1989; Malim et al., 1989b; Malim et al., 1990; Olsen et al., 1990; Bartel et al., 1991; Huang et al., 1991; Dayton et al., 1992; Holland et al., 1992; Mann et al., 1994). For full activity, further Rev monomers must be able to oligomerize along stem I of the RRE (Heaphy et al., 1990, 1991; Malim & Cullen, 1991; Mann et al., 1994). Truncations of Stem I that do not affect the high-affinity motif reduce Rev responses by removing additional potential binding sites for Rev monomers, with the longest truncations producing the greatest losses of activity (Mann et al., 1994). Similarly, mutations in the Rev protein that block oligomerization along the RNA stem result in an inactive protein (Malim & Cullen, 1991; Zapp et al., 1991).
It has been suggested that up to twelve Rev monomers in total can bind to each wild-type RRE (Mann et al., 1994). The high-affinity motif is not the sole Rev binding site on the RRE, however, unless a monomer is bound to the high-affinity motif, the oligomerization of Rev cannot take place. The binding of a single Rev to the high-affinity motif facilitates the binding and co-operative oligomerization of additional Rev monomers along the RRE (Iwai et al., 1992; Mann et al., 1994), with neighbouring Rev monomers in contact with one another (Mann et al., 1994).
Various models have been proposed as to the mechanism by which Rev oligomerization is achieved. Kjems et al. (1991) suggested that Rev monomers bind to a variety of sequence-specific sites in the RRE. Zapp et al. (1991) argued that Rev binds to the RRE high-affinity site as a pre-existing tetramer. Malim & Cullen (1991) ascribed the oligomerization solely to protein/protein interactions between neighbouring Rev monomers, and Tiley et al. (1992) reached the same conclusion. Powell et al. (1995) refined this view, believing that sequence-specific information in the RNA can exert a subtle influence on higher-order binding, but maintain that protein/protein interactions are the major determinant directing oligomerization.
Disruption of the natural Rev/RRE interaction via mutation of the natural sequences has been explored in the prior art as a potential avenue to the use of altered Rev or RRE molecules in anti-HIV therapy. Transdominant Rev mutants which retain the RRE-binding features of wild-type Rev but which are defective in certain other features have been described(eg. Malim et al., 1989a; Malim et al., 1991; Bogerd et al., 1995).
Harada et al. (1996) relates to in vivo methods for selecting short peptides which bind Rev.
Jensen, K. B. et al. (1995) disclose chemically modified RNA sequences (i.e., containing 5-iodouridine) which bind Rev in vitro with higher affinity than the RRE and which are able to crosslink with Rev at a 1:1 ratio. These are postulated as potential suicide ligands for in vivo disease inhibition, however, non-specific interactions with chemically reactive bases cannot be ruled out in an in vivo situation.
WO92/05195 discloses molecules which mimic the high-affinity binding site of the native RRE in order to act as competitive inhibitors, thus sequestering free Rev protein and preventing it from interacting with those mRNAs which contain the RRE. These molecules contain a greater number of Rev binding sites than are contained in viral RRE-containing mRNAs.
One object of the invention is to provide nucleic acid molecules which inhibit HIV replication.
Another object of the invention is to provide a nucleic acid decoy which binds HIV Rev protein so as to inhibit HIV infection.
Another object of the invention is to provide a nucleic acid decoy which binds HIV Rev protein with greater co-operativity than the wild-type RRE.